Nisin compositions for use as enhanced, broad range bactericides

ABSTRACT

Bacteriocin compositions comprising lanthionine containing bacteriocins and non-bactericidal agents. When the bacteriocin compositions are combined with a suitable carrier with each component present in sufficient quantities such that the composition is effective against Gram negative bacteria in addition to Gram positive bacteria, they become enhanced, rapid acting, broad range bactericides suitable for a variety of applications.

This application is a divisional of application Ser. No. 07/870,803,filed on Apr. 17, 1992, now U.S. Pat. No. 5,260,271 which is acontinuation of application Ser. No. 07/317,626, filed on Mar. 1, 1989(abandoned), which is a continuation-in-part of application Ser. No.07/209,861, filed on Jun. 22, 1988 (abandoned).

BACKGROUND OF THE INVENTION

Nisin is a polypeptide with antimicrobial properties which is producedin nature by various strains of the bacterium Streptococcus lactis. Itis a known food preservative which inhibits the outgrowth of spores ofcertain species of Gram positive Bacilli.

Although sometimes mistakenly and imprecisely referred to as anantibiotic, nisin is more correctly classified as a bacteriocin, i.e. aproteinaceous substance produced by bacteria and which has antibacterialactivity only towards species closely related to the species of itsorigin. Nisin is a naturally-occurring preservative found in lowconcentration in milk and cheese, and is believed to be completelynon-toxic and non-allergenic to humans.

Nisin has recently been recognized as safe by the FDA as a direct foodingredient in pasteurized cheese spread, pasteurized processed cheesespread, and pasteurized or pasteurized processed cheese spread withfruits, vegetables, or meats. Furthermore, since it is a polypeptide,any nisin residues remaining in foods are quickly digested.

A summary of nisin's properties appears in Hurst, A., Advances inApplied Microbiology 27:85-123 (1981). This publication describes whatis generally known about nisin. Nisin, produced by Streptococcus lactis,is available commercially as an impure preparation, Nisaplin™, fromAplin & Barrett Ltd., Dorset, England and can be obtained by isolatingnaturally-occurring nisin from cultures of Streptococcus lactis and thenconcentrating the nisin according to known methods. There are alsoreported methods for producing nisin using altered strains ofStreptococcus. See Gonzalez et al., U.S. Pat. No. 4,716,115, issued Dec.29, 1987. It should also be possible to produce nisin by recombinant DNAtechnology.

Nisin has been applied effectively as a preservative in dairy products,such as processed cheese, cream and milk. The use of nisin in processedcheese products has been the subject of recent patents. See U.S. Pat.Nos. 4,584,199 and 4,597,972. The use of nisin to inhibit the growth ofcertain Gram positive bacteria has been well documented. However, itscomplete success and acceptance as a food preservative has heretoforebeen hampered by the belief that nisin was ineffective against Gramnegative and many Gram positive bacteria. Gram negative bacteria arealmost always present in conjunction with Gram positive bacteria and area major source of food spoilage and contamination. See Taylor, U.S. Pat.No. 5,584,199, issued Apr. 22, 1986 and Taylor, U.S. Pat. No. 4,597,972,issued Jul. 1, 1986; Tsai and Sandine, "Conjugal Transfer of NisinPlasmid Genes from Streptococus Lactis 7962 to Leuconostoc Dextranicum181, Applied and Environmental Microbiology, Feb. 1987, p. 352; "ANatural Preservative," Food Engineering Int'l,, May 1987, pp. 37-38;"Focus on Nisin," Food Manufacture, March 1987, p. 63.

SUMMARY OF THE INVENTION

It has now been found that contrary to prior teaching, compositionscomprising nisin, in combination with various non-bactericidal agentshave enhanced, broad range bactericidal activity against Gram negativebacteria as well as enhanced activity against a broader range of Grampositive bacteria than nisin alone. The enhanced bactericidal activityagainst Gram positive bacteria occurs in a pH range broader thanpreviously taught. The invention provides bacteriocin compositions ofnisin or other, lanthionine containing bacteriocins, in combination withvarious non-bactericidal agents for example chelating agents orsurfactants. The invention further provides the compositions dissolvedor suspended in a suitable carrier to yield enhanced broad rangebactericides.

DETAILED DESCRIPTION OF THE INVENTION

Specifically, it has been found that a solution of about 0.1 μg/ml to300 μg/ml of nisin in the presence of about 0.1 mM to 20 mM of achelating agent, for example EDTA, virtually eliminates the growth ofGram negative bacteria such as Salmonella typhimurium, Escherichia coli,Pseudomonas aeruginosa, Bacterioides gingivalis, Actinobacillusactinomycetescomitans, and Klebsiella pneumoniae and is more activetowards Gram positive bacteria such as Staphylococcus aureus,Streptococcus mutans, Listeria monocytogenes Streptococcus agalactiaeand Coryneform bacteria than nisin alone. Although the enhancement ofnisin activity by chelator was concentration dependent, contrary toexpectations, concentrations of EDTA in excess of 20 mM were inhibitoryto the bactericidal activity of nisin. However, in the presence of aproteinaceous carrier, and polyvalent polymers such as serum albumin,collagen, gelatin, casein and keratin, the inhibition of nisin byconcentrations of EDTA above 20 mM was significantly reduced, therebyextending the useful range of EDTA enhancement of nisin.

It has also been found that a solution of about 0.1 μg/ml to 300 μg/mlnisin and about 0.1 mM to 20 mM of a chelating agent will furtherenhance the effectiveness of nisin against Gram negative and Grampositive bacteria in the presence of about 0.01% to 1.0% of surfactant.Additionally, it has been found that, in the presence of surfactantalone, nisin has enhanced activity against Gram positive bacteria.

In the present invention, suitable chelating agents include, but are notlimited to, EDTA, CaEDTA, CaNa₂ EDTA, and other alkyldiaminetetraacetates, EGTA and citrate. Surfactants, valuable as cleansingagents, suitable for combination with nisin, with or without EDTA,include, but are not limited to, the nonionic surfactants Tweens,Tritons, and glycerides, ionic surfactants such as fatty acids,quaternary compounds, anionic surfactants such as sodium dodecylsulphate and amphoteric surfactants such as cocamidopropyl betaine andemulsifiers.

Since Gram positive and Gram negative bacteria are almost always foundtogether in foods, the effectiveness of the nisin compositions towardsGram negative bacteria such as Salmonella typhimurium, Escherichia Coli,Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacterioides gingivalis,Actinobacillus actinomycetescomitans, and other Gram negative pathogensand Gram positive bacteria will be of great use. The bactericides areparticularly suited for the control and prevention of contamination ofraw ingredients, processed foods and beverages by bacterial pathogensand other microbial spoilage organisms. Potential food related usesinclude treatment of meats, especially poultry, eggs, cheese and fishand treatment of food packaging and handling equipment. Further usesinclude as food preservative, such as in processed cheese, cream, milk,dairy products and in cleaning poultry, fish, meats, vegetables, anddairy and food processing equipment. The use of the nisin compositionsshould not be limited to food related uses and the nisin compositionsshould be useful in any situation in which there is a need or desire toeliminate Gram negative and Gram positive bacteria.

The compositions can be dissolved in a suitable carrier for example anaqueous solvent or buffer or suspended in any suitable liquid, colloidalor polymeric matrix to create bactericides. The compositions orbactericides can be incorporated into ointments or coatings formedicinal uses such as the treatment of infections, wound dressings orsurgical implants and as a broad spectrum disinfectant for skin or oralrinses, disinfectant scrubs, wipes or lotions. The bactericides can beused for cleaning medical instruments, in pre-operative surgical scrubsand the like. The bactericides are particularly useful in circumstanceswhere environmental disinfection is desired but where chemicalgermicidals are precluded because of the risks of corrosive or otherwisetoxic residues.

Unlike the activity of most broad spectrum germicidals which iscompromised by the presence of complex organic matter, the compositionsof the present invention are effective as bactericides in the presenceof organic matter, such as milk or serum.

Nisin was known to optimally inhibit the growth of a few closely relatedGram positive bacteria, particularly certain Gram positive spore formingbacilli at pH 5.0. The bactericidal activity of nisin in solution with achelating agent was surprisingly rapid and greatly enhanced towards abroad range of Gram positive bacteria at pH values greater than pH 5.0,and, moreover, was activated towards Gram negative bacteria at bothacidic and basic pH, preferably in the range pH 5.0 to 8.0. Thisunexpectedly rapid and broad-ranged bactericidal activity ofchelator-activated nisin makes it suitable for use as, among otherthings, a disinfectant.

Nisin belongs to the class of peptide bacteriocins containinglanthionine. Also included among that class are subtilin, epidermin,cinnamycin, duramycin, ancovenin and Pep 5. These bacteriocin peptidesare each produced by different microorganisms. However, subtilinobtained from certain cultures of Bacillus subtilis, and epiderminobtained from certain cultures of Staphylococcus epidermis, have beenfound to have molecular structures very similar to that of nisin (seeHurst, pp. 85-86, and Schnell et al., Nature, 333:276-278). It istherefore believed that because of the molecular similarities, otherlanthionine containing peptide bacteriocins will be equally as effectiveas nisin in combination with chelating agents and non-ionic surfactantsin eliminating Gram negative and Gram positive bacterial contaminations.

The effectiveness of the nisin, and by extension other lanthioninecontaining peptide bacteriocin, compositions as bactericides againstGram negative bacteria is surprising, since the prior art generallyteaches away from this activity of nisin. The enhanced activity of nisinagainst Gram positive bacteria in the presence of EDTA at a pH greaterthan 5.0 is unexpected since it was previously believed that nisinactivity is optimal at pH 5.0. Furthermore, the discovery of sucheffectiveness of the nisin and lanthionine containing peptidebacteriocin compositions as bactericides fulfills a long-felt need inthe science of food preservation, which has suffered from the absence ofan acceptable, natural, non-toxic agent effective against a broad rangeof bacteria.

In order to demonstrate the superior and unexpected rapid activity ofthe composition containing nisin, EDTA and/or various surfactantsagainst both Gram negative and Gram positive bacteria, a number ofexperiments were conducted with the bactericides. These experiments aremeant as illustration and are not intended to limit this invention. Itis to be expected that other, lanthionine containing peptidebacteriocins would be effective substitutes for nisin and that chelatingagents other than EDTA will be effective substitutes for EDTA.

All tests in the following examples were performed at 37° C. Theefficacy of the enhanced broad range bactericides was determined byassaying bactericidal activity as measured by the percent bacterialsurvival after treatment with the bactericide. Generally, afterincubation of a 10⁷ cell per ml suspension of target species with thenovel bactericide for specified lengths of time, bacteria were collectedby centrifugation for 2 minutes. The bacterial pellet was washed free ofthe bactericide with a rescue buffer, termed herein Phage buffer (50 mMTris-HCl buffer pH 7.8, 1 mM MgSO₄, 4 mM CaCl₂, 0.1 M Nacl, and 0.1%gelatin), resuspended and serially diluted into Phage buffer, and 100 μlof the suspended bacteria were spread on nutrient agar plates. Survivingbacteria were determined by scoring colony forming units (CFU) afterincubation for 24-48 hours at 37° C. An effective bactericide accordingto this invention is one which allows less than 0.1% of the initialviable count of the bacteria to survive.

EXAMPLE 1 Activity of Nisin and a Chelating Agent Against Gram NegativeBacteria (S. typhimurium)

As shown in Table 1, two tests were conducted in 20 mM Tris, pH 8.0 at37° C. to show the effect of the bactericide containing nisin and thechelating agent EDTA alone. Test #1, a control, was conducted withoutEDTA and shows the effect of nisin alone toward the Gram negativebacterium S. typhimurium. The increased concentrations of nisin doexhibit some activity, but even the activity of the higherconcentrations in the absence of EDTA, 1.6% survival at 100 μg/ml nisin,is wholly inadequate for a food preservative. The level of bactericidalactivity obtained from nisin and EDTA is significant.

                  TABLE 1                                                         ______________________________________                                                      Nisin (μg/ml)                                                              0    10     30     50  100  130                                 Test Initial Viable                                                                           EDTA    Percentage S. typhimurium                             #    Bacteria Count                                                                           (mM)    Survival at 3 hours                                   ______________________________________                                        1    3.0 × 10.sup.6                                                                      0      100  51.3 --   7.0 1.6  --                            2    5.7 × 10.sup.6                                                                     20      2.5  --   10.sup.-3                                                                          --  <10.sup.-4                                                                         <10.sup.-4                    ______________________________________                                    

Test #2 (Table 1), conducted using nisin plus 20 mM EDTA, demonstratesthe surprising activity of the nisin composition in eliminating thetarget Gram negative bacteria.

Table 1 shows that in test #2 at a concentration of 20 mM EDTA and 30μg/ml of nisin, the bactericide has a marked bactericidal activitytowards S. typhimurium, while at nisin concentrations of 100 μg/ml andgreater, the nisin and EDTA bactericide virtually eliminates thebacteria (percentage survival less than 10⁻⁴ which indicates nosurviving bacteria in the assay). Thus, the combination of EDTA andnisin demonstrates a synergistic activity of greater than 1000 timesthat of nisin alone.

EXAMPLE 2 Activity of Nisin, a Chelating Agent and a Surfactant AgainstGram Negative Bacteria (S. typhimurium)

Four tests (Table 2) were conducted to determine the effect on S.typhimurium of the bactericide containing nisin and both EDTA and thesurfactant Triton X-100 in 20 mM Tris, pH 8.0 at 37°. The control (Test#1) is identical to the control of Example 1 (Table 1).

                                      TABLE 2                                     __________________________________________________________________________                      Nisin (μg/ml)                                                          Triton                                                                            0   10 30  50 100 300                                       Test                                                                             Initial Viable                                                                       EDTA                                                                              X-100                                                                             Percentage S. typhimurium                                   #  Bacteria Count                                                                       (mM)                                                                              (%) Survival at 3 hours                                         __________________________________________________________________________    1  3.0 × 10.sup.6                                                                  0  0   100 51.3                                                                             --   7.0                                                                              1.6                                                                              --                                        2  3.0 × 10.sup.6                                                                  0  1.0 37.4                                                                              93.0                                                                             --  64.0                                                                             47.0                                                                              --                                        3  5.7 × 10.sup.6                                                                 20  0.1 0.03                                                                              -- <10.sup.-3                                                                        -- --  --                                        4  5.7 × 10.sup.6                                                                 20  1.0 <10.sup.-4                                                                        -- <10.sup.-4                                                                        -- <10.sup.-4                                                                        <10.sup.-4                                __________________________________________________________________________

Test #2 (Table 2) was conducted using nisin and 1.0% Triton X-100, butwithout EDTA. The presence of the detergent alone inhibits the activityof the nisin towards the Gram negative bacteria and nisin wasineffective. However, in tests #3 and #4 (Table 2), which represent theinvention, the presence of 20 mM EDTA in combination with Triton X-100is a bactericide which markedly increases the bactericidal activity ofnisin towards S. typhimurium. Indeed the combination of Triton X-100With EDTA but without nisin was effective, although to a lesser degreethan in the presence of nisin. While in both tests #3 and #4 (Table 2)the nisin combinations were very effective, the concentration of 1.0%Triton X-100 (test #4, Table 2) was most effective.

The presence of the non-ionic surfactant, Triton X-100, in combinationwith EDTA, enhances the activity of nisin toward Gram negative bacteriaeven more than the bactericide containing nisin and EDTA alone (Example1).

EXAMPLE 3

Activity of Nisin, a Chelating Agent and a Surfactant Against GramNegative Bacteria (S. typhimurium)

Table 3 shows the enhanced activity toward S. typhimurium of thebactericide containing nisin, 20 mM of the chelating agent EDTA and thenon-ionic surfactant Tween 20 in 20 mM Tris, pH 8.0 at 37° C. As withTriton X-100 (Example 2) the combination of nisin and EDTA with (I%) ofTween 20 is most effective.

                                      TABLE 3                                     __________________________________________________________________________                       Nisin (μg/ml)                                                              0   10 30  50                                                                              100 300                                       Test                                                                             Initial Viable                                                                       EDTA                                                                              Tween 20                                                                           Percentage S. typhimurium                                  #  Bacteria Count                                                                       (mM)                                                                              (%)  Survival at 3 hours                                        __________________________________________________________________________    1  3.0 × 10.sup.6                                                                  0  0    100 51.3                                                                             --  7.0                                                                             1.6 --                                        2  5.7 × 10.sup.6                                                                 20  0    2.5 -- <10.sup.-3                                                                        --                                                                              <10.sup.-4                                                                        <10.sup.-4                                3  4.3 × 10.sup.6                                                                 20  1.0  <10.sup.-2                                                                        -- <10.sup.-4                                                                        --                                                                              <10.sup.-4                                                                        <10.sup.-4                                __________________________________________________________________________

EXAMPLE 4 Activity of Nisin, a Chelating Agent and a Surfactant AgainstGram Negative Bacteria (Escherichia coli)

The effect of the bactericide containing nisin and EDTA towards the Gramnegative bacteria E. coli was demonstrated, as shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                           Nisin (μg/ml)                                                         Triton 0      30     100  300                                   Test Initial Viable                                                                           EDTA    X-100  Percentage E. coli                             #    Bacterial Count                                                                          (mM)    (%)    Survival at 2 hours                            ______________________________________                                        1    1.0 × 10.sup.7                                                                      0      0      100  27   25   8.5                             2    1.0 × 10.sup.7                                                                     20      0      14.5 0.86 0.01 0.001                           3    1.0 × 10.sup.7                                                                      0      1.0    100  --   30   --                              4    1.0 × 10.sup.7                                                                     20      1.0    1.2  0.8  0.05 <10.sup.-4                      ______________________________________                                    

The tests, with and without EDTA, were performed in 20 mM Tris buffersolution pH 8.0 at 37° C. with an initial viable count of 1×10⁷ E. colicells/ml. The effects of the bactericide were measured as a function ofpercentage bacteria survival after 2 hours.

In test #1, (control, Table 4) without EDTA, nisin exhibited littlemeaningful activity toward the elimination of E. coli. In test #2 (Table4), however, where 20 mM EDTA was present, the bactericidal compositionexhibited substantial activity towards the E. coli bacteria. Theactivity increased in effectiveness as the concentration of nisin wasincreased. The combination of nisin with EDTA as a bactericidedemonstrates a 1000 fold synergistic increase in effectiveness towardsE. coli. In tests #3 and #4 (Table 4), it can be seen that Triton X-100has no significant bactericidal activity towards E. coli. In fact,Triton X-100 appears to inhibit nisin activity towards Gram negativebacteria as was found with S. typhimurium (Table 2). However, theoverall enhancement of nisin by EDTA substantially reverses theinhibitory effects of Triton X-100 as seen in Tables 2 and 4.

It thus appears that the bactericide containing nisin and a chelatingagent, such as EDTA, is an effective food preservative towards varioustypes of Gram negative bacteria even in the presence of surfactants.

EXAMPLE 5 Activity of Nisin and a Chelating Agent Against Gram NegativeBacteria (Klebsiella pneumoniae)

The effect of the bactericide containing nisin and EDTA alone towardsthe Gram negative bacteria K. pneumoniae was demonstrated, as shown inTable 5.

                  TABLE 5                                                         ______________________________________                                                      Triton                                                                              Nisin μg/ml                                            Initial Viable                                                                            EDTA    X-100   0    30   100  300                                Test Bacteria Count                                                                           (mM)    (%)   % Survival at 2 hours                           ______________________________________                                        1    10.sup.7    0      0     100  --   50   38                               2    10.sup.7   20      0      22  0.5  1.1  0.085                            ______________________________________                                    

The two tests, one with and one without EDTA (control), were performedin 20 mM Tris buffer, pH 8.0 at 37° C. with an initial viable count of10⁷ cells/ml of K. pneumoniae. The effect was measured as a function ofpercentage bacterial survival after 2 hours.

In test #1, (control, Table 5) without EDTA, nisin exhibited littlemeaningful bactericidal activity toward K. pneumoniae. In test #2 (Table5), however, where 20 mM EDTA was present, the bactericide exhibitedsubstantial activity towards K. pneumoniae. The activity increased ineffectiveness as the concentration of nisin was increased.

EXAMPLE 6 Nisin Activity Against Gram Negative Bacteria (Salmonellatyphimurium) is Dependent on Chelator Concentration

The data in Table 6 demonstrate that the enhanced activation of nisintowards Gram negative bacteria (S. typhimurium) is dependent on theconcentration of EDTA in either 50 mM sodium acetate, pH 5.0, or 20 mMTris, pH 8.0 at 37° C.

                                      TABLE 6                                     __________________________________________________________________________                     EDTA (mM)                                                    Test  Initial Viable                                                                       Nisin                                                                            0    0.2                                                                              2.0 10  50  100                                       #  pH Bacterial Count                                                                      μg/ml                                                                          % Survival at 2 hours                                        __________________________________________________________________________    1  5.0                                                                              3 × 10.sup.6                                                                    0 100  -- 38.7                                                                              15.2                                                                              3.5 --                                        2  5.0                                                                              3 × 10.sup.6                                                                   100                                                                              0.6  10.sup.-4                                                                        10.sup.-4                                                                         0.004                                                                             0.02                                                                              --                                        3  8.0                                                                              5 × 10.sup.6                                                                    0 100  --  8.7                                                                              14  11.4                                                                              45                                        4  8.0                                                                              5 × 10.sup.6                                                                   100                                                                              4    10.sup.-4                                                                        10.sup.-4                                                                         10.sup.-4                                                                         0.6 30                                        __________________________________________________________________________

In tests #1 and #3, (controls, Table 6) using EDTA concentrations up to100 mM without nisin exhibited little meaningful activity towards S.typhimurium at either pH 5.0 (#1) or pH 8.0 (#3). In tests #2 and #4(Table 6), however, where 100 μg/ml nisin was present in combinationwith EDTA, the bactericides exhibited substantial activity towards S.typhimurium. The activity of the bactericides was similar at both acidicpH (5.0) and basic pH (8.0), despite the fact that the activity of nisinalone towards Gram positive bacteria is optimal at pH 5.0.

The enhancement of nisin by EDTA was concentration dependent, beingoptimal in the range 0.2 mM to 10 mM at pH values 5.0 and 8.0.Surprisingly, at concentrations greater than 10 mM EDTA, the enhancementof nisin by EDTA becomes reduced; the reduction of activation issignificantly greater at pH 8.0 than at pH 5.0.

EXAMPLE 7 Nisin and a Chelating Agent Against Gram Negative Bacteria (S.typhimurium)

The enhancement of the activity of nisin by EDTA towards Gram negativebacteria in the presence of biological tissue was demonstrated with S.typhimurium on chicken muscle, and is shown in Table 7.

                                      TABLE 7                                     __________________________________________________________________________             EDTA (mM)                                                            Test  Nisin                                                                            0   0.1                                                                              0.3 1  3   10 20 30 100                                       #  pH μg/ml                                                                         % Survival.sup.a at 2 hours                                          __________________________________________________________________________    1  5.0                                                                               0 11.8                                                                              -- --  -- --  6.4                                                                              -- -- --                                        2  5.0                                                                              300                                                                              0.1 0.2                                                                              0.05                                                                              0.01                                                                             0.003                                                                             0.016                                                                            0.03                                                                             0.02                                                                             0.07                                      3  8.0                                                                               0 100 -- --  -- --  5.2                                                                              -- -- --                                        4  8.0                                                                              300                                                                              7.5 0.1                                                                              0.02                                                                              0.02                                                                             0.09                                                                              0.47                                                                             0.5                                                                              -- 2.2                                       5  8.0                                                                              .sup. 300.sup.b                                                                  0.02                                                                              0.09                                                                             0.0002                                                                            <10.sup.-4                                                                       0.0004                                                                            0.003                                                                            -- 0.03                                                                             0.09                                      __________________________________________________________________________     .sup.a Unadhered cells                                                        .sup.b Contains 1% Bovine serum albumin (BSA)                            

Incubations were performed in either 50 mM sodium acetate, pH 5.0, or 20mM Tris, pH 8.0 at 37° C.

Cubes of chicken muscle were cleansed with sodium hypochlorite andpovidone iodine prior to use. To inoculate the tissue, the cubes ofchicken muscle were dipped into a 10⁸ cells/ml suspension of S.typhimurium in 20 mM Tris HCl, pH 8.0. Excess moisture was removed fromdipped cubes by tapping. The chicken samples were placed into sufficientbuffer containing the nisin composition to cover the tissue andincubated for 2 hours at 37° C. after which the tissue was removed tosufficient Phage buffer to cover the tissue. The bacteria remaining inthe test solution were collected by centrifugation, washed with Phagebuffer, and combined with bacteria washed from the tissue by Phagebuffer. The combined samples (termed "unadhered" cells) were seriallydiluted and 100 μl aliquots were plated for determination of survivingbacteria.

In tests #1 and #3 (Table 7), in the absence of nisin at either pH 5 orpH 8, EDTA alone has no significant effect on the survival of S.typhimurium. In tests #2 and #4 (Table 7), however, where 300 μg/mlnisin was present, the bactericides exhibited substantial activitytowards S. typhimurium on chicken muscle at both pH 5.0 and pH 8.0.

The enhancement of nisin by EDTA was concentration dependent, theoptimal concentration being in the range 0.3 mM to 10 mM EDTA at both pHvalues 5.0 and 8.0. At concentrations greater than 10 mM EDTA at pH 8.0,the activation of nisin by EDTA is reduced. However, as is shown in test#5 (Table 7), in the presence of 1.0% bovine serum albumin at pH 8.0,the efficacy of nisin towards S. typhimurium on chicken muscle isexpressed throughout the range of EDTA concentrations up to 100 mM.

Thus, bactericides containing nisin and low concentrations of chelatingagent, such as EDTA in the range 0.1 mM to 20 mM, can be extremelyeffective for the elimination or prevention of contamination of food byGram negative bacteria.

EXAMPLE 8 Titration of Nisin Activity Against Gram Negative Bacteria (S.typhimurium)

At the optimal concentration of chelating agent, the efficacy of thebactericide in Tris buffer towards Gram negative bacteria wasdemonstrated to be substantial, as is shown in Table 8.

                                      TABLE 8                                     __________________________________________________________________________                     Nisin μg/ml                                               Test                                                                             Initial Viable                                                                       EDTA                                                                              BSA                                                                              0  0.1                                                                             0.3                                                                              1.0                                                                              3.0                                                                              10 30 100                                      #  Bacterial Count                                                                      (mM)                                                                              %  % Survival at 2 hours                                        __________________________________________________________________________    1  6 × 10.sup.6                                                                   0   0  100                                                                              --                                                                              -- -- -- 51.3                                                                             -- 1.6                                      2  6 × 10.sup.6                                                                   1.0 1.0                                                                               63                                                                              0.7                                                                             0.08                                                                             0.01                                                                             0.05                                                                             0.01                                                                             <10.sup.-4                                                                       --                                       __________________________________________________________________________

In test #2 (Table 8), it can be seen that as little as 0.3 μg/ml ofnisin, with 1.0 mM EDTA in 20 mM Tris at pH 8.0 in the presence of 1%bovine serum albumin (BSA), significantly reduced the survival of S.typhimurium. The bactericide is as active towards Gram negative bacteriaas nisin alone is towards Gram positive Streptococci.

EXAMPLE 9 Titration of Nisin Activity Against Gram Negative Bacteria (S.typhimurium)

At the optimal concentration of chelating agent, the efficacy of abactericide towards Gram negative bacteria in the presence of biologicaltissue was demonstrated with S. typhimurium on chicken muscle, and isshown in Table 9.

                                      TABLE 9                                     __________________________________________________________________________                          Nisin μg/ml                                          Test   Initial Viable                                                                       EDTA                                                                              BSA 0  10  100                                                                              200 300                                       #  pH  Bacterial Count                                                                      (mM)                                                                              (%) % Survival at 2 hours                                   __________________________________________________________________________    1  8.0 3 × 10.sup.7                                                                   0   0   100                                                                              --  -- --  --                                        2  8.0 3 × 10.sup.7                                                                   1.0 1.0  27                                                                              0.26                                                                              0.008                                                                            0.007                                                                             0.006                                     __________________________________________________________________________

Cubes of chicken muscle were cleansed with sodium hypochlorite andpovidone iodine prior to use. To inoculate the tissue, the cubes ofchicken muscle were dipped into a 10⁸ cells/ml suspension of S.typhimurium in 20 mM Tris HCl, pH 8.0. Excess moisture was removed fromdipped cubes by tapping. The tissue was placed into sufficient buffercontaining the nisin compositions to cover the tissue, and incubated for2 hours at 37° C. after which the tissue was removed to sufficient Phagebuffer to cover the tissue. The bacteria remaining in the test solutionwere collected by centrifugation, washed with Phage buffer, and combinedwith bacteria washed from the tissue by Phage buffer. The combinedsamples (termed "unadhered" cells) were serially diluted and 100 μlaliquots were plated for determination of surviving bacteria.

EXAMPLE 10 Nisin EDTA and Methyl Paraben Activity Against Gram NegativeBacteria (S. typhimurium)

A bactericide containing nisin and EDTA, when combined with a known foodpreservative, methyl paraben, was demonstrated to be exceptionallyeffective towards Gram negative bacteria, as shown in Table 10.

                  TABLE 10                                                        ______________________________________                                                            % Methyl Paraben                                          Test  Initial Viable                                                                           Nisin  EDTA.sup.b                                                                            0    0.1    1.0                               #     Bacterial Count                                                                          μg/ml                                                                             (mM)    % Survival.sup.c at 2 hours                   ______________________________________                                        1     3 × 10.sup.6                                                                        0     10      11.8 1.0      10.sup.-4                       2     3 × 10.sup.6                                                                       300    10      0.03 <10.sup.-3                                                                           <10.sup.-4                        ______________________________________                                         .sup.b 50 mM Na acetate buffer, pH 5.0                                        .sup.c Unadhered cells                                                   

Cubes of chicken muscle were cleaned with sodium hypochlorite andpovidone iodine prior to use. To inoculate the tissue, the cubes ofchicken muscle were dipped into a 10⁸ cells/ml suspension of S.typhimurium in 50 mM sodium acetate buffer, pH 5.0. Excess moisture wasremoved from dipped cubes by tapping. The tissue was placed intosufficient buffer containing nisin compositions to cover the tissue, andincubated for 2 hours at 37° C. after which the tissue was removed tosufficient Phage buffer to cover the tissue. The bacteria remaining inthe test solution were collected by centrifugation, washed with Phagebuffer, and combined with bacteria washed from the tissue by Phagebuffer. The combined samples (termed "unadhered" cells) were seriallydiluted and 100 μl aliquots were plated for determination of survivingbacteria.

In test #1 (Table 10), methyl paraben in the presence of 10 mM EDTA wasshown to be effective towards S. typhimurium only at a concentration of1.0%. In test #2 (Table 10), however, in the presence of 300 μg/mlnisin, the effectiveness of methyl paraben and nisin towards S.typhimurium was substantially improved.

The compositions containing nisin and EDTA significantly improve theutility of the food preservative methyl paraben. Furthermore, thebactericides may lead to substantial reductions in the concentrations,or eliminate the need for these commonly recognized, though lessdesirable, food preservatives such as methyl paraben.

EXAMPLE 11 Nisin and Chelating Agent Activity Against Gram PositiveBacteria (Staphylococcus aureus)

The activation of nisin by a chelating agent is pH-dependent. The datain Table 11 confirm that at pH 5.0, nisin is somewhat more bactericidaltowards S. aureus than is nisin at pH 8.0. At pH 5.0, EDTA does notenhance nisin activity towards S. aureus and at concentrations of EDTAgreater than 10 mM, EDTA is inhibitory to the bactericidal activity ofnisin. However, the bactericidal activity of nisin activated by EDTA atpH 8.0 is significantly greater than the bactericidal activity of nisinalone, or in combination with EDTA at pH 5.0.

                  TABLE 11                                                        ______________________________________                                        Influence of pH on the Effects of EDTA on Nisin                               Bactericidal Activity towards Staphyloccus aureus                             EDTA mM                                                                            Nisin  0      0.1  0.3   1.0  3.0  10   30   100                         pH   μg/ml                                                                             % Survival 2 hr.sup.a                                             ______________________________________                                        8.0  0      100    --   100   81   100  100  100  --                          8.0  3.0    7.4    0.03 0.01  0.2  0.4   3    56  --                          5.0  0      100    --   --    --   100  --   --   --                          5.0  3.0    0.6    1.0  1.3   1.4  1.8  --    34  80                          ______________________________________                                         .sup.a Initial viable count: 8.0 × 10.sup.6 cfu/ml                      Incubations were performed in 50 mM sodium acetate buffer, pH 5.0 or 20 m     TrisHCl buffer, pH 8.0 at 37°0 C.                                 

The bactericidal activity of nisin alone is reported (see Hurst) to begreatest at pH 5.0 or lower, and data presented in Table 11 supportthis. On the basis of this information it was believed that thebactericidal activation of nisin by EDTA towards S. aureus wouldlikewise be greatest at lower pH. However, as can be seen in Table 11and contrary to expectations (see Table 6), EDTA was not observed toenhance nisin activity towards Gram positive bacteria at pH 5.0.However, inhibition of nisin activity by high concentrations of EDTA wasstill observed at pH 5.0. Thus, the activation of nisin by a chelatingagent occurs only within a range of chelator concentrations and, withrespect to Gram positive bacteria, is dependent upon pH with thepreferred pH range greater than pH 5.0.

EXAMPLE 12 Nisin and Chelating Agent Activity Against Gram PositiveBacteria

The effects of EDTA on the bactericidal activity of nisin at pH 8.0 arenot limited to S. aureus, an important human pathogen, but are alsoobserved with Streptococcus mutans, responsible for dental plaque (Table12A), Listeria monocytogenes, a foodborne pathogen (Table 12B), and witha mixed population of axillary Coryneform bacteria, contributors to bodyodor (Table 12C).

                  TABLE 12A                                                       ______________________________________                                        The Effects of EDTA on the Bactericidal                                       Activity of Nisin towards Streptococcus mutans                                EDTA mM                                                                       Nisin  0      0.01   0.1  0.3  1.0  3.0  10  30   100                         pH  μg/ml                                                                             % Survival after 2 hr.sup.a                                        ______________________________________                                        8.0 0      100    --   --   --   --   --   --  --   --                        8.0 0.1    4.3    1.8  0.04 0.02 0.06 1    25  100  100                       ______________________________________                                         .sup.a Initial viable count: 6.0 × 10.sup.6 cfu/ml                      Incubations were performed in 20 mM TrisHCl, pH 8.0 at 37° C.     

                  TABLE 12B                                                       ______________________________________                                        The Effects of EDTA on the Bactericidal                                       Activity towards Listeria monocytogenes                                       EDTA mM                                                                       Nisin   0       0.1    0.3  1.0  3.0  10   30   100                           pH  μg/ml                                                                              % Survival after 2 hr.sup.a                                       ______________________________________                                        8.0 0       100     --   --   84   --   --   --   --                          8.0 3.0     0.71    0.04 0.04 0.02 0.1  0.64 10   14                          ______________________________________                                         .sup.a Initial viable count: 6.0 × 10.sup.6 cfu/ml                      Incubations were performed in 20 mM TrisHCl, pH 8.0 at 37° C.     

                  TABLE 12C                                                       ______________________________________                                        The Effects of EDTA on Nisin Bactericidal                                     Activity towards Coryneform bacteria                                          EDTA mM                                                                       Nisin   0         0.1    0.3     1.0  3.0   10                                pH   μg/ml                                                                             % Survival 2 hr.sup.a                                             ______________________________________                                        8.0  0      100       --   4.6     3.6  8     36                              8.0  3      0.22      0.03 0.0009  0.1  --    0.16                            ______________________________________                                         .sup.a Initial viable count: 1.0 × 10.sup.6 cfu/ml                      Incubations were performed in 20 mM TrisHCl, pH 8.0 at 37° C.     

EXAMPLE 13 Rapid Bactericidal Activity of Nisin Activated by Chelator

The bactericide comprising nisin with EDTA is rapidly bactericidal as isillustrated by the data presented in Table 13A. Suspensions of the Grampositive bacterium S. mutans at 10⁷ cells/ml were incubated in 20 mMTris buffer, pH 7.3 at 37° C. with a range of concentrations of nisinactivated by 1 mM EDTA. The suspensions were incubated for various timesranging from 0.5 to 60 minutes with the bactericides. The bactericidalefficacy of the bactericides was estimated by determining the percentsurvival of bacteria. Enhanced by EDTA, as little as 10 μg/ml of thenisin in this formulation is able to reduce the bacterial load by 6 logswithin 1 minute.

Rapid bactericidal activity is a prerequisite for effectivedisinfection. Thus, the compositions are predicted to be effectivebactericides particularly as demonstrated here, as a component of amouthwash, rinse, toothpaste, or other similar dentrifice active againstplaque forming S. mutans.

The activity of nisin enhanced by EDTA against Gram negative bacteriaafter 2-3 hours was shown in Examples 1-7. Rapid bactericidal activityof nisin enhanced by EDTA is also seen towards Gram negative bacteriaand this is illustrated by the data in Table 13B.

                  TABLE 13A                                                       ______________________________________                                        Kinetics of Bactericidal Activity towards                                     Streptococcus mutans of Nisin Enhanced by EDTA                                Incubation                                                                             Nisin μg/ml with 1.0 mM EDTA                                      Time     0      1        3    10     30   100                                 (Minutes)                                                                              % Survival.sup.a                                                     ______________________________________                                        0.5      --     --       --   --     --   <10.sup.-4                          1        --     --       --   <10.sup.-4                                                                           <10.sup.-4                                                                         <10.sup.-4                          3        100    0.5      0.002                                                                              <10.sup.-4                                                                           <10.sup.-4                                                                         --                                  15       --     0.03     <10.sup.-4                                                                         <10.sup.-4                                                                           --   --                                  30       --     --       <10.sup.-4                                                                         --     --   --                                  60       100    0.003    --   --     --   --                                  ______________________________________                                         .sup.a Control viable cell count: 1.0 × 10.sup.7 cfu/ml                 Incubations were performed in 20 mM TrisHCl, pH 7.3 at 37° C.     

                  TABLE 13B                                                       ______________________________________                                        Rapid Bactericidal Activity towards Escherichia coli                          of Nisin Enhanced by EDTA                                                     Nisin μg/ml                                                                0          0.3      1.0   3      10   30    100                               mM EDTA % survival at 1 min.sup.a                                             ______________________________________                                        1.0     100    100      56  0.37   0.013                                                                              0.015 0.008                           ______________________________________                                         .sup.a Initial viable count: 1.0 × 10.sup.7 cfu/ml                      Incubations were performed in 20 mM Tris, pH 7.0 at 37° C.        

EXAMPLE 14 Effect of Divalent Cations on EDTA Enhancement of NisinActivity

Divalent cations bind to EDTA and other chelating agents and would beexpected to neutralize the activation of nisin by EDTA. However, as canbe seen by the data in Table 14, the bactericidal activity of nisinagainst S. mutans is enhanced by 1 mM EDTA even in the presence of 1 mMCa²⁺ ion; only above 3 mM was Ca²⁺ ion inhibitory to EDTA-activatednisin. This is particularly important in mouthwash applications wherecalcium ion concentrations are relevant.

                  TABLE 14                                                        ______________________________________                                        Rapid Bactericidal Activity towards Streptococcus mutans                      of Nisin Activated by EDTA in the presence of Divalent Cation                 CaCl.sub.3 mM                                                                 Nisin 0        0.1      0.3    1.0    3      10                               ______________________________________                                        % survival at 1 min..sup.a                                                    0     100                                                                     3     2.9                                                                     3.sup.E                                                                             0.0042   0.0042          0.052         18                               30.sup.E                                                                            0.0019            0.0003 0.0004 0.06   6.8                              100.sup.E                                                                           <10.sup.-4        <10.sup.-4                                                                           <10.sup.-4                                                                           0.0001 1.5                              ______________________________________                                         .sup.E 1 mM Na.sub.2 EDTA                                                     .sup.a Initial viable count 1.0 × 10.sup.2 cfu/ml.                      Incubations performed in 10% Fetal Calf Serum at 37° C.           

EXAMPLE 15 Nisin and Surfactant Activity Against Gram Positive Bacteria

The bactericidal activity of nisin can also be significantly enhancedwhen combined with a surfactant alone. This is best illustrated at alimiting nisin concentration (0.2 μg/ml) as shown in Table 15A. Atconcentrations up to 0.1%, the food grade surfactant monolaurin haslittle significant bactericidal activity towards Streptococcusagalactiae in the complex medium milk. Nisin, at concentrations up to0.2 μg/ml, likewise does not exhibit significant bactericidal activityin milk. However, the combination of the two agents, 0.1% monolaurin andnisin 0.2 μg/ml, is extremely potent towards S. agalactiae. Thisbactericide is over 100 times more active than what would be expectedfor the additive effect and 10,000 times more active than either of thecomponents individually. Thus, when the application of nisin is limitedby its available activity, a bactericide comprising nisin with asurfactant can be expected to be more useful.

An example of where the application of nisin is limited by its availableactivity is illustrated by the data in Table 15B. Although nisin, andparticularly the bactericide comprising nisin and EDTA, is bactericidaltowards L. monocytogenes, the data in Table 15B demonstrate that in acomplex medium like milk the available nisin activity towards thisorganism is restricted. However, the bactericide comprised of nisin withthe glyceride, monooleate, is effective in milk towards this foodbornepathogen even though monooleate by itself had no bactericidal activitytowards this organism.

                  TABLE 15A                                                       ______________________________________                                        Nisin Bactericidal Activity towards                                           Streptococcus agalactiae in milk at 37° C.                             (Activation of nisin by monolaurin)                                                        Monolaurin                                                       Nisin        (%)                                                              (μg/ml)   0          0.01      0.1                                         ______________________________________                                                   % survival at 2h.sup.a                                             0            100        100       4.5                                         0.02         100        100       0.2                                         0.2          2.2        0.05      0.0008                                      ______________________________________                                         .sup.a Initial viable counts 6.0 × 10.sup.7 cfu/μl.                  Incubations were in milk at 37° C.                                

                  TABLE 15B                                                       ______________________________________                                        Nisin Bactericidal Activity towards                                           Listeria monocytogenes in milk at 37° C.                               (Activation of nisin by monooleate)                                           Nisin        % Monooleate                                                     (μg/ml)   0          0.1       1.0                                         ______________________________________                                                   % Survival 2hr.sup.a                                                0           100        67        63                                          100          0.56       10.sup.-3 10.sup.-4                                   ______________________________________                                         .sup.a Initial viable counts 5.0 × 10.sup.7 cfu/ml                      Incubations were in milk at 37° C.                                

We claim:
 1. A bactericidal composition consisting essentially of alanthionine-containing bacteriocin and a surfactant.
 2. The compositionas defined in claim 1 wherein the lanthionine-containing bacteriocin isselected from the group consisting of nisin, subtilin, epidermin,cinnamycin, duramycin, ancovenin and Pep
 5. 3. The composition asdefined in claim 1 wherein the surfactant is selected from the groupconsisting of fatty acids, amphoteric surfactants and nonionicsurfactants.
 4. An enhanced broad range bactericide consistingessentially of a lanthionine-containing bacteriocin, a surfactant and acarrier.
 5. The composition of claim 1 or 4 wherein the surfactant is anemulsifier.
 6. The enhanced broad range bactericide as in claim 4wherein the lanthionine-containing bacteriocin is selected from thegroup consisting of nisin, subtilin, epidermin, cinnamycin, duramycin,ancovenin and Pep 5 and the surfactant is selected from the groupconsisting of fatty acids, amphoteric surfactants and nonionicsurfactants and wherein the bacteriocin and surfactant are present inamounts sufficient such that the bactericide has enhanced effectivenessagainst at least one of the bacteria from the group consisting of Gramnegative and Gram positive bacteria.
 7. The enhanced broad rangebactericide as in claim 6 wherein the concentration of bacteriocin isbetween about 0.1 μg/ml and 300.0 μg/ml and the concentration ofsurfactant is between about 0.01% and 1.0%.
 8. The composition of claim3 or 6 wherein the nonionic surfactant is a glyceride.
 9. Thecomposition of claim 3 or 6 wherein the amphoteric surfactant is aquaternary compound.
 10. A method of obtaining enhanced bactericidalactivity of a lanthionine-containing bacteriocin which comprisescombining the bacteriocin with a surfactant.
 11. The method of claim 10wherein the lanthionine containing bacteriocin is nisin.